The present invention is related to non-nucleoside inhibitors of reverse transcriptase (RT). In particular, the present invention relates to a novel class of substituted benzimidazoles effective in the inhibition of human immunodeficiency virus (HIV) RT.
Since its recognition in 1981, the acquired immunodeficiency syndrome (AIDS) has become a major pandemic. The worldwide prevalence of HIV infection has been estimated at more than 18,500,000 cases, with an additional estimate of 1.5 million infected children (R. Famighetti, 1996 World Almanac and Book of Facts, World Almanac Books, Mahwah, N.J., [1995], p. 840).
The etiologic agent associated with AIDS was identified as the human immunodeficiency virus (HIV). HIV is classified as a retrovirus, as it contains reverse transcriptase (RT), a multi-functional enzyme that contains RNA-dependent DNA polymerase activity, as well as DNA-dependent DNA polymerase and ribonuclease H activities. These three activities are essential for the conversion of genomic retroviral RNA into double-stranded DNA that can then be integrated into an infected host cell genome.
HIV is a D-type virus within the lentivirus family, with two major antigenic types (HIV-1 and HIV-2). HIV-1 and HIV-2 share approximately 40% genetic identity, although they can be readily distinguished based on differences in antibody reactivity to the envelope glycoprotein (M. Cloyd, xe2x80x9cHuman Retroviruses,xe2x80x9d in S. Baron (ed.), Medical Microbiology, University of Texas Medical Branch at Galveston, [1996], pp. 761-775). Both HIV-1 and HIV-2 have been associated with AIDS.
The search for effective drugs against HIV has focused on targeting various critical components of the replication cycle of HIV-1. One important component in this cycle is the reverse transcriptase enzyme. Indeed, perhaps because of its pivotal role in the life cycle of HIV, it was the target of the first clinically approved anti-retroviral agents (see, Patel et al., xe2x80x9cInsights into DNA Polymerization Mechanisms from Structure and Function Analysis of HIV-1 Reverse Transcriptase,xe2x80x9d Biochem., 34:5351-5363 [1995]), although other compounds such as protease inhibitors have recently been introduced. In addition to its critical role in HIV replication, targeting RT has a potential benefit in reducing the toxicity to the patient associated with many drugs, as human cells do not normally contain this RT activity. Therefore, the potential for targeted inhibition of only viral replication and not host cell multiplication is present. However, this potential has yet to be realized.
There are two major classes of RT inhibitors. The first comprises nucleoside analogues, such as 3xe2x80x2-azido-3xe2x80x2-deoxythymidine (AZT), 2xe2x80x2,3xe2x80x2-didehydro-2xe2x80x2,3xe2x80x2-dideoxythymidine (d4T), and 2xe2x80x2,3xe2x80x2-dideoxycytidine (ddC). These compounds are analogs of normal deoxynucleoside triphosphates (dNTPs). However, these are not specific for HIV RT, and are incorporated into cellular DNA by host DNA polymerases; thus, these compounds can cause serious side effects. Moreover, administration of these analogs has resulted in the emergence of drug-resistant viral strains that contain mutations in their RT. Thus, these RT inhibitors have dangers that must be considered in developing treatment regimens for HIV-infected patients.
The second major class of RT inhibitors comprises the non-nucleoside RT inhibitors (NNRTI), such as tetrahydroimidazo(4,5,1-1-jk)(1,4)-benzodiazepin-2-(1H)-one, and -thione (TIBO) derivatives, dipyridodiazepinones, pyridinones, bis(heteroaryl)piperazines (BHAPs), 2xe2x80x2,5xe2x80x2-bis-O-(tertbutyldimethylsilyl)-3xe2x80x2-spiro-5xe2x80x3-(4xe2x80x3-amino-1xe2x80x3,2xe2x80x3-oxathiole-2xe2x80x3,2xe2x80x3-dioxide)pyrimidine (TSAO) derivatives, xcex1-anilinophenylacetamide (xcex1-APA), 8-chloro-4,5,6,7-tetrahydro-5-methylimidazo-[4,5,1-jk][1,4]benzodiazepine-2 (1)-one (8-Cl TIBO), and nevirapine. (See, e.g. Pauwels et al., xe2x80x9cPotent and Selective Inhibition of HIV-1 Replication in Vitro By a Novel Series of TIBO Derivatives,xe2x80x9d Nature 343:470-474 [1990]; Merluzzi et al., xe2x80x9cInhibition of HIV-1 Replication by a Non-Nucleoside Reverse Transcriptase Inhibitor,xe2x80x9d Science 250:1411-1413 [1990]; Goldman and Stern, xe2x80x9cPyridinone Derivatives: Specific Human Immunodeficiency Virus Type 1 Reverse Transcriptase Inhibitors with Antiviral Activity,xe2x80x9d Proc. Natl. Acad. Sci. USA 88:6863-6867 [1991]; Romero and Tarpley, xe2x80x9cNon-Nucleoside Reverse Transcriptase Inhibitors that Potently and Specifically Block Human Immunodeficiency Virus Type 1 Replication,xe2x80x9d Proc. Natl. Acad. Sci., USA 88:8806-8810 [1991]; Balzarini et al., xe2x80x9c2xe2x80x2,3xe2x80x2-Bis-O-(Tertbutyldimethylsilyl)-3xe2x80x2-Spiro-5xe2x80x3-(4xe2x80x3-Amino-1xe2x80x3,2xe2x80x3-Oxathiole-2xe2x80x3,2xe2x80x3-Dioxide) Pyrimidine (TSAO) Nucleoside Analogs: Highly Selective Inhibitors of Human Immunodeficiency Virus Type 1 That are Targeted at the Viral Reverse Transcriptase,xe2x80x9d Proc. Natl. Acad. Sci. USA 89:4392-4396 [1992]; Young, xe2x80x9cNon-Nucleoside Inhibitors of HIV-1 Reverse Transcriptase,xe2x80x9d Perspect. Drug Discov. Des., 1:181-192; and Pauwels et al., xe2x80x9cPotent and Highly Selective Human Immunodeficiency Virus Type 1 (HIV-1) Inhibition by a Series of xcex1-Anilinophenylacetamide Derivatives Targeted at HIV-1 Reverse Transcriptase,xe2x80x9d Proc. Natl. Acad. Sci., USA 90:1711-1715 [1993]).
Unlike the nucleoside analogues, the NNRTIs do not act as chain terminators and do not bind at the dNTP-binding site. The majority of these compounds have been shown to share a common binding site unique to HIV-1 RT that is located in proximity to the RT polymerase active site. (See, Tantillo et al., xe2x80x9cLocations of Anti-AIDS Drug Binding Sites and Resistance Mutations in Three-Dimensional Structure of HIV-1 Reverse Transcriptase. Implications for Mechanisms of Drug Inhibition and Resistance,xe2x80x9d J. Mol. Biol., 243:369-387 [1994]; Smith et al., xe2x80x9cMolecular Modeling Studies of HIV-1 Reverse Transcriptase Nonnucleoside Inhibitors: Total Energy of Complexation as a Predictor of Drug Placement and Activity,xe2x80x9d Prot. Sci., 4:2203-2222 [1995]; Ding et al., xe2x80x9cStructure of HIV-1 TR/TIBO R86183 Complex Reveals Remarkable Similarity in the Binding of Diverse Nonnucleoside Inhibitors,xe2x80x9d Nature Struct. Biol., 2:407-415 [1995]; and Nanni et al., xe2x80x9cReview of HIV-1 Reverse Transcriptase Three Dimensional Structure: Implications for Drug Design,xe2x80x9d Perspect. Drug Discov. Des., 1:129-150 [1993]).
NNRTIs are highly specific for HIV-1 RT, and do not inhibit either HIV-2 RT or normal cellular polymerases, resulting in lower cytotoxicity and fewer side effects than the nucleoside analogs. (See, e.g., Ding et al., xe2x80x9cStructure of HIV-1 Reverse Transcriptase in a Complex with the Non-Nucleoside Inhibitor xcex1-APA R 95845 at 2.8 xc3x85 Resolution,xe2x80x9d Structure 3:365-379 [1995]). However, resistance to some of these compounds has been reported. (See, e.g., Nunberg et al., xe2x80x9cViral Resistance to Human Immunodeficiency Virus Type 1-Specific Pyridinone Reverse Transcriptase Inhibitors,xe2x80x9d J. Virol., 65:4887-4892 [1991]; Tantillo et al., xe2x80x9cLocations of Anti-AIDS Drug Binding Sites and Resistance Mutations in the Three-Dimensional Structure of HIV-1 Reverse Transcriptase: Implications for Mechanisms of Drug Inhibition and Resistance,xe2x80x9d J. Mol. Biol., 243:369-387; and Richman, xe2x80x9cResistance of Clinical Isolates of Human Immunodeficiency Virus to Antiretroviral Agents,xe2x80x9d Antimicrob. Agents Chemother., 37:1207-1213 [1993]).
Despite recent developments in drug and compound design to combat HIV, there remains a need for a potent, non-toxic compound that is effective against wild type (WT) RTs, as well as RTs that have undergone mutations, and thereby become refractory to commonly used anti-HIV compounds.
The present invention is related to substituted benzimidazole compounds. In particular, the present invention provides non-nucleoside inhibitors of reverse transcriptase (RT) comprising a novel class of substituted benzimidazoles effective in the inhibition of human immunodeficiency virus (HIV) RT.
In one embodiment, the present invention provides substituted benzimidazoles having the structure of FIG. 7A. In one embodiment, Xxe2x80x3 is selected from the group consisting of hydrogen, methyl, ethyl, cyano, methoxyl, nitro, amine, acetamide, methylamine, dimethylamine, isopropyl, isopropenyl, bromine and chlorine. In another embodiment, Rxe2x80x3 is selected from the group consisting of 2,6-difluorobenzyl (2,6-F2Bn), benzyl (Bn), ethylbenzyl, 2,6-dichlorobenzyl (2,6-Cl2Bn), 2,3,4,5,6-pentafluorobenzyl (2,3,4,5,6-F5Bn), pyridylmethyl (CH2(3-Py), benzenesulfonyl (PhSO2), 2,6-difluorobenzoyl (2,6-F2Bz), and 3,3-dimethylallyl. In yet another embodiment, Xxe2x80x3 is selected from the group consisting of hydrogen, methyl, ethyl, cyano, methoxyl, nitro, amine, acetamide, methylamine, dimethylamine, isopropyl, isopropenyl, bromine and chlorine; and Rxe2x80x3 is selected from the group consisting of 2,6-difluorobenzyl, benzyl, ethylbenzyl, 2,6-dichlorobenzyl, 2,3,4,5,6-pentafluorobenzyl, pyridylmethyl, benzenesulfonyl, 2,6-difluorobenzoyl, and 3,3-dimethylallyl. In a preferred embodiment, Rxe2x80x3 is 2,6-difluorobenzyl.
In an alternative embodiment, the present invention provides substituted benzimidazoles with the general structure of FIG. 7B. In one embodiment, Xxe2x80x2 is selected from the group consisting of hydrogen, methyl, ethyl, cyano, methoxyl, nitro, amine, acetamide, methylamine, dimethylamine, isopropyl, isopropenyl, bromine and chlorine. In another embodiment, Rxe2x80x2 is selected from the group consisting of phenyl (Ph), formyl (CHO), isopropyl (iPr), H, methyl (CH3), cyclopropyl, hydroxymethyl (CH2OH), and 2,6-difluorobenzyloxymethyl (CH2O(2,6-F2Bn), 2,6 difluorophenyl (2,6-F2Ph), methylphenyl (2-CH3Ph), 2-fluoro-6-methoxylphenyl, pyridyl (e.g., 4-Py, 3-Py), and naphthyl (e.g., 1-Nap, 2-Nap). In yet another embodiment, Xxe2x80x2 is selected from the group consisting of hydrogen and methyl, ethyl, cyano, methoxyl, nitro, amine, acetamide, methylamine, dimethylamine, isopropyl, isopropenyl, bromine and chlorine; and Rxe2x80x2 is selected from the group consisting of phenyl, formyl, isopropyl, H, methyl, cyclopropyl, hydroxymethyl, 2,6-difluorobenzyloxymethyl, 2,6 difluorophenyl (2,6-F2Ph), methylphenyl (2-CH3Ph), 2-fluoro-6-methoxylphenyl, pyridyl (e.g., 4-Py, 3-Py), and naphthyl (e.g., 1-Nap, 2-Nap). In a preferred embodiment, Rxe2x80x2 is 2,6-difluorophenyl.
In another alternative embodiment, the present invention provides substituted benzimidazoles with the general structure of FIG. 13A, wherein Xxe2x80x3xe2x80x3 is at least one substituent positioned at a benzimidazole carbon of a substituted 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole and selected from C4, C5, C6, C7, C4 and C5, and C4 and C6 of the substituted 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole, and wherein said Xxe2x80x3xe2x80x3 is selected from the group consisting of hydrogen, 4-methyl, 5-methyl, 6-methyl, 7-methyl, 4,5-dimethyl, 4,6-dimethyl, 4-ethyl, 4-cyano (CN), 4-methylenecyano (CH2CN), 4-methoxy, 4-nitro, 5-nitro, 4-amino, 4-N-acetamido (NHAc), 4-N-methylamino (NHCH3), 4-N-dimethylamino [N(CH3)2], 4-isopropyl, 4-isoprenyl, 4-bromo, 5-bromo, 6-bromo, 4-chloro, 5-chloro, 6-chloro, fluoro, N-methylacetamido (NAcCH3), methyl carboxylate (CO2Me), methyl acetamido ester (CH2OAc), hydroxy, CNH(NH2OH), amide (CONH2), N-dimethylamide [CON(CH3)2], carboxylic acid, hydroxymethyl (CH2OH), formyl, chloromethyl, 4-methyleneazido (CH2N3), aminomethyl (CH2NH2), N-acetamidomethyl (CH2NHAc), CH2NCH2Ac, dimethyl ether (CH2OCH3), 4-methylenethiocyanate (CH2NCS), and 4-methyleneacetylene (CH2CCH). In a preferred embodiment, Xxe2x80x3xe2x80x3 is selected from the group consisting of hydrogen, 4-methyl, 4-ethyl, 4-cyano, 4-methoxy, 4-nitro, 4-amino, 4-N-acetamido, 4-N-methylamino, 4-N-dimethylamino, 4-isopropyl, 4-isopropenyl, 4-bromo, 4-chloro, 4-methyleneazido, 4-methylenecyano, 4-methylenethiocyanate, and 4-methyleneacetylene. The present invention also provides pharmaceutical compositions of substituted benzimidazoles having the general structure of FIG. 13A, wherein Xxe2x80x3xe2x80x3 is selected from the group consisting of hydrogen, 4-methyl, 4-ethyl, 4-cyano, 4-methoxy, 4-nitro, 4-amino, 4-N-acetamido, 4-N-methylamino, 4-N-dimethylamino, 4-isopropyl, 4-isopropenyl, 4-bromo, 4-chloro, 4-methyleneazido, 4-methylenecyano, 4-methylenethiocyanate, and 4-methyleneacetylene.
It is contemplated that the substituted benzimidazoles of the present invention comprise derivatives containing various groups. It is not intended that the present invention be limited to particular substituted benzimidazole derivatives. For example, it is intended that the present invention encompasses embodiments in which such groups as aromatic rings, hydrocarbons, and other structures are included. Such groups include, but are not limited to hydrogen, 4-methyl, 5-methyl, 6-methyl, 7-methyl, 4,5-dimethyl, 4,6-dimethyl, 4-chloro, 5-chloro, 6-chloro, 4-bromo, 5-bromo, 4-nitro, and 5-nitro, cyclopropyl, 2,6-difluorobenzyloxymethyl, 2,6-difluorophenyl, 2-fluoro-6-methoxyphenyl, pyridyl, naphthyl, 2,6-difluorobenzyl, benzyl, ethylbenzyl, 2,6-dichlorobenzyl, 2,3,4,5,6-pentafluorobenzyl, pyridylmethyl, benzenesulfonyl, 2,6-difluorobenzoyl, 3,3-dimethylallyl, fluoro, chloromethyl, methoxy, N-methylacetamide (NAcCH3), ethyl, methyl carboxylate (CO2Me), methyl acetamido ester (CH2OAc), hydroxy, amine, N-acetamide (NHAc), N-methylamine (NHCH3), N-diethylamine [N(CH3)2], cyano, CNH(NH2OH), amide (CONH2), N-dimethylamide [CON(CH3)2], carboxylic acid, isopropyl, isoprenyl, hydroxymethyl (CH2OH), formyl, chloromethyl, aminomethyl (CH2NH2), N-acetamidomethyl (CH2NHAc), CH2NCH2Ac, dimethyl ether (CH2OCH3), 4-methyleneazido (CH2N3), 4-methylenecyano, 4-methylenethiocyanate, and 4-methyleneacetylene. It is further intended that these groups be included in these compositions alone or in combination.
It is also contemplated that the aromatic residues of various embodiments of the present invention may be replaced with hydrophobic residues, such as aliphatic groups. For example, the present invention encompasses alkylimidazoles, including, but not limited to 1-(2,6-difluorobenzyl)-2-difluorophenyl-5,6-dialkylimidazole.
It is further contemplated that the present invention includes embodiments in which the carbons (C) present on the benzyl ring (i.e., C4, C5, C6, and C7) are replaced with nitrogen (N), singly, or in combination (e.g., azapurines).
It is contemplated that the substituted benzimidazoles of the present invention will find use in the treatment of HIV infection/disease. In particularly preferred embodiments, the present invention provides pharmaceutical compositions comprising substituted benzimidazoles with activity against HIV-1 RT.
The present invention also provides methods for treating human immunodeficiency virus (HIV) infection, comprising the steps of: a) providing: i) a subject suspected of being infected with human immunodeficiency virus; and ii) a composition having anti-reverse transcriptase activity and comprising at least one substituted benzimidazole having a 2,6-difluorobenzyl substituent at the N-1 site of the benzimidazole ring; b) exposing the subject to the composition having anti-reverse transcriptase activity; and c) observing for inhibition of anti-reverse transcriptase activity. In one preferred embodiment, the human immunodeficiency virus is HIV-1.
In one embodiment, the methods for treating HIV infection utilize substituted benzimidazoles further comprising a 2,6-difluorophenyl substituent at the C2 site of the benzimidazole ring. In particularly preferred embodiments, the methods for treating HIV infection utilize substituted benzimidazoles having the structure of FIG. 13A, wherein Xxe2x80x3xe2x80x3 is at least one substituent positioned at a benzimidazole carbon selected from C4, C5, C6, C7, C4 and C5, and C4 and C6 of a substituted 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole, and wherein said Xxe2x80x3xe2x80x3 is selected from the group consisting of hydrogen, 4-methyl, 5-methyl, 6-methyl, 7-methyl, 4,5-dimethyl, 4,6-dimethyl, 4-ethyl, 4-cyano (CN), 4-methylenecyano (CH2CN), 4-methoxy, 4-nitro, 5-nitro, 4-amino, 4-N-acetamido (NHAc), 4-N-methylamino (NHCH3), 4-N-dimethylamino [N(CH3)2], 4-isopropyl, 4-isoprenyl, 4-bromo, 5-bromo, 6-bromo, 4-chloro, 5-chloro, 6-chloro, fluoro, N-methylacetamido (NAcCH3), methyl carboxylate (CO2Me), methyl acetamido ester (CH2OAc), hydroxy, CNH(NH2OH), amide (CONH2), N-dimethylamide [CON(CH3)2], carboxylic acid, hydroxymethyl (CH2OH), formyl, chloromethyl, 4-methyleneazido (CH2N3), aminomethyl (CH2NH2), N-acetamidomethyl (CH2NHAc), CH2NCH2Ac, dimethyl ether (CH2OCH3), 4-methylenethiocyanate (CH2NCS), and 4-methyleneacetylene (CH2CCH). In preferred embodiments, Xxe2x80x3xe2x80x3 is selected from the group consisting of hydrogen, 4-methyl, 4-ethyl, 4-cyano, 4-methoxy, 4-nitro, 4-amino, 4-N-acetamido, 4-N-methylamino, 4-N-dimethylamino, 4-isopropyl, 4-isopropenyl, 4-bromo, 4-chloro, 4-methyleneazido, 4-methylenecyano, 4-methylenethiocyanate, and 4-methyleneacetylene.
In another embodiment, the methods for treating human immunodeficiency virus infection utilize substituted benzimidazoles having the structure of FIG. 7A, wherein Xxe2x80x3 is selected from the group consisting of hydrogen, methyl, ethyl, cyano, methoxyl, nitro, amine, acetamide, methylamine, dimethylamine, isopropyl, isopropenyl, bromine and chlorine; and Rxe2x80x3 is selected from the group consisting of 2,6-difluorobenzyl, benzyl, ethylbenzyl, 2,6-dichlorobenzyl, 2,3,4,5,6-pentafluorobenzyl, pyridylmethyl, benzenesulfonyl, 2,6-difluorobenzoyl, and 3,3-dimethylallyl. In a preferred embodiment, Rxe2x80x3 is 2,6-difluorobenzyl. In yet another embodiment, the methods for treating human immunodeficiency virus infection utilize substituted benzimidazoles having the structure of FIG. 7B, wherein Xxe2x80x2 is selected from the group consisting of hydrogen and methyl, ethyl, cyano, methoxyl, nitro, amine, acetamide, methylamine, dimethylamine, isopropyl, isopropenyl, bromine and chlorine; and Rxe2x80x2 is selected from the group consisting of phenyl, formyl, isopropyl, H, methyl, cyclopropyl, hydroxymethyl, 2,6-difluorobenzyloxymethyl, 2,6 difluorophenyl (2,6-F2Ph), methylphenyl (2-CH3Ph), 2-fluoro-6-methoxylphenyl, pyridyl (e.g., 4-Py, 3-Py), and naphthyl (e.g., 1-Nap, 2-Nap). In a preferred embodiment, Rxe2x80x2 is 2,6-difluorophenyl.
Furthermore, the present invention provides methods for inhibiting HIV-1 reverse transcriptase, comprising the steps of: a) providing: i) a sample suspected of being infected with HIV-1 virus; and ii) a composition having anti-reverse transcriptase activity and comprising at least one substituted benzimidazole having a 2,6-difluorobenzyl substituent at the N-1 site of said benzimidazole; b) exposing the sample to the composition having anti-reverse transcriptase activity; and c) observing for inhibition of said anti-reverse transcriptase activity.
In some embodiments, the methods for inhibiting HIV-1 reverse transcriptase utilize substituted benzimidazoles further comprising a 2,6-difluorophenyl substituent at the C2 site of the benzimidazole ring. In particularly preferred embodiments, the methods for inhibiting HIV-1 reverse transcriptase utilize substituted benzimidazoles having the structure of FIG. 13A, wherein Xxe2x80x3xe2x80x3 is at least one substituent positioned at a benzimidazole carbon selected from C4, C5, C6, C7, C4 and C5, and C4 and C6 of a substituted 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole, and wherein said Xxe2x80x3xe2x80x3 is selected from the group consisting of hydrogen, 4-methyl, 5-methyl, 6-methyl, 7-methyl, 4,5-dimethyl, 4,6-dimethyl, 4-ethyl, 4-cyano (CN), 4-methylenecyano (CH2CN), 4-methoxy, 4-nitro, 5-nitro, 4-amino, 4-N-acetamido (NHAc), 4-N-methylamino (NHCH3), 4-N-diinethylamino [N(CH3)2], 4-isopropyl, 4-isoprenyl, 4-bromo, 5-bromo, 6-bromo, 4-chloro, 5-chloro, 6-chloro, fluoro, N-methylacetamido (NAcCH3), methyl carboxylate (CO2Me), methyl acetamido ester (CH2OAc), hydroxy, CNH(NH2OH), amide (CONH2), N-dimethylamide [CON(CH3)2], carboxylic acid, hydroxymethyl (CH2OH), formyl, chloromethyl, 4-methyleneazido (CH2N3), aminomethyl (CH2NH2), N-acetamidomethyl (CH2NHAc), CH2NCH2Ac, dimethyl ether (CH2OCH3), 4-methylenethiocyanate (CH2NCS), and 4-methyleneacetylene (CH2CCH). In preferred embodiments, Xxe2x80x3xe2x80x3 is selected from the group consisting of hydrogen, 4-methyl, 4-ethyl, 4-cyano, 4-methoxy, 4-nitro, 4-amino, 4-N-acetamido, 4-N-methylamino, 4-N-dimethylamino, 4-isopropyl, 4-isopropenyl, 4-bromo, 4-chloro, 4-methyleneazido, 4-methylenecyano, 4-methylenethiocyanate, and 4-methyleneacetylene.
In another embodiment, the methods for inhibiting HIV-1 reverse transcriptase utilize substituted benzimidazoles having the structure of FIG. 7A, wherein Xxe2x80x3 is selected from the group consisting of hydrogen, methyl, ethyl, cyano, methoxyl, nitro, amine, acetamide, methylamine, dimethylamine, isopropyl, isopropenyl, bromine and chlorine; and Rxe2x80x3 is selected from the group consisting of 2,6-difluorobenzyl, benzyl, ethylbenzyl, 2,6-dichlorobenzyl, 2,3,4,5,6-pentafluorobenzyl, pyridylmethyl, benzenesulfonyl, 2,6-difluorobenzoyl, and 3,3-dimethylallyl. In a preferred embodiment, Rxe2x80x3 is 2,6-difluorobenzyl. In yet another embodiment, the methods for inhibiting HIV-1 reverse transcriptase utilize substituted benzimidazoles having the structure of FIG. 7B, wherein Xxe2x80x2 is selected from the group consisting of hydrogen and methyl, H, methyl, ethyl, cyano, methoxyl, nitro, amine, acetamide, methylamine, dimethylamine, isopropyl, isopropenyl, bromine and chlorine; and Rxe2x80x2 is selected from the group consisting of phenyl, formyl, isopropyl, H, methyl, cyclopropyl, hydroxymethyl, 2,6-difluorobenzyloxymethyl, 2,6 difluorophenyl (2,6-F2Ph), methylphenyl (2-CH3Ph), 2-fluoro-6-methoxylphenyl, pyridyl (e.g., 4-Py, 3-Py), and naphthyl (e.g., 1-Nap, 2-Nap). In a preferred embodiment, Rxe2x80x2 is 2,6-difluorophenyl.
In addition, it is contemplated that the present invention encompasses analogs of the benzimidazole ring system which are capable of undergoing dissociation in the binding pocket of HIV RT, to give rise to electrophilic intermediates that react with nucleophilic sites in the pocket. Thus, it is contemplated that compounds that act as irreversible inhibitors of HIV RT also be encompassed as embodiments within the present invention.
It is not intended that the compounds of the present invention be limited to any particular use. Indeed, it is intended that the compounds of the present invention will be utilized against organisms other than HIV.
Definitions
To facilitate understanding of the invention, a number of terms are defined below.
As used herein, the term xe2x80x9cretrovirusxe2x80x9d refers to a group of viruses with RNA genomes. Retroviruses are characterized as having reverse transcriptase, the enzyme that allows the RNA genome to be transcribed into DNA.
As used herein, the term xe2x80x9creverse transcriptasexe2x80x9d refers to an enzyme with RNA-dependent DNA polymerase activity, with or without the usually associated DNA-dependent DNA polymerase and ribonuclease activity observed with wild-type reverse transcriptases.
As used herein, the term xe2x80x9canti-viralxe2x80x9d is used in reference to any compound, substance, or molecule capable of inhibiting or preventing viral replication and/or dissemination. It is intended that the term encompasses compounds capable of inhibiting viral replication by interfering with such activities as the reverse transcriptase activity of retroviruses. It is also intended to encompass xe2x80x9cnon-nucleoside reverse transcriptase inhibitorsxe2x80x9d (NNRTI). In preferred embodiments, the term is used in reference to substituted benzimidazole compounds.
As used herein, the term xe2x80x9cchemotherapeuticxe2x80x9d refers to any compound, element, or substance useful against disease. In preferred embodiments, the term encompasses compounds such as the substituted benzimidazoles of the present invention.
As used herein, the term xe2x80x9cpurifiedxe2x80x9d refers to the removal of contaminants from a sample. Methods such as carbon, hydrogen and nitrogen analyses (CHN analysis, or xe2x80x9celemental analysisxe2x80x9d) may be used to determine the purity of compounds. In preferred embodiments, the CHN values of compounds of the present invention are very close to the predicted values. Correspondence of experimental with the predicted values to within 0.3% indicates high levels of purity. In particularly preferred embodiments, the compounds of the present invention have CHN values within 0.3% of the predicted values. In less preferred embodiments, the level of purity may be lower (ie., greater than 0.3% difference between the predicted and actual CHN values).
As used herein, the terms xe2x80x9cbenzimidazolexe2x80x9d and xe2x80x9csubstituted benzimidazolexe2x80x9d are used in reference to molecules with the core structure as indicated in FIG. 1. It is intended that the term encompasses compounds in which substitutions, including additions, have been made to the chemical structure. The term encompasses, but is not limited to substitution reactions, wherein there is replacement of one or more atoms or group in a molecule by another atom or group. In a preferred embodiment, the present invention encompasses 1- and 2-substituted benzimidazoles (See e.g., FIG. 7), including but not limited to 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (See e.g., FIG. 13).
As used herein, the term xe2x80x9csubstituentxe2x80x9d is used in reference to functional and molecular groups present in the substituted benzimidazole. The terms xe2x80x9cC2,xe2x80x9d xe2x80x9cC4,xe2x80x9d xe2x80x9cC5,xe2x80x9d xe2x80x9cC6,xe2x80x9d xe2x80x9cC7xe2x80x9d, etc. refer to a carbon in the benzimidazole ring and its position in the benzimidazole ring, following the numbering convention for the benzimidazole ring as indicated in FIG. 1. For example, the term dependent xe2x80x9cC4 and C5xe2x80x9d refers to the carbons at the 4 and 5 positions of the benzimidazole ring, while the term xe2x80x9cC4 and C6xe2x80x9d refers to carbons at the 4 and 6 positions of the benzimidazole ring.
As used herein, the term xe2x80x9cTBZxe2x80x9d refers to 1-(2,6-difluorophenyl)-1H,3-thiazolo[3,4-a]benzimidazole.
The term xe2x80x9ccyclic compoundsxe2x80x9d refers to compounds having one (i.e., a monocyclic compounds) or more than one (i.e., polycyclic compounds) ring of atoms. The term is not limited to compounds with rings containing a particular number of atoms. While most cyclic compounds contain rings with five or six atoms, rings with other numbers of atoms (e.g., three or four atoms) are also contemplated by the present invention. The identity of the atoms in the rings is not limited, though the atoms are usually predominantly carbon atoms. Generally speaking, the rings of polycyclic compounds are adjacent to one another; however, the term xe2x80x9cpolycyclicxe2x80x9d compound includes those compounds containing multiple rings that are not adjacent to each other.
The term xe2x80x9cheterocyclic compoundsxe2x80x9d refers broadly to cyclic compounds wherein one or more of the rings contains more than one type of atom. In general, carbon represents the predominant atom, while the other atoms include, for example, nitrogen, sulfur, and oxygen. Examples of heterocyclic compounds include benzimidazole, furan, pyrrole, thiophene, and pyridine.
The terms xe2x80x9caromatic,xe2x80x9d xe2x80x9caromatic compounds,xe2x80x9d and the like refer broadly to compounds with rings of atoms having delocalized electrons. The monocyclic compound benzene (C6H6) is a common aromatic compound. However, electron delocalization can occur over more than one adjacent ring (e.g., naphthalene [two rings] and anthracene [three rings]). Different classes of aromatic compounds include, but are not limited to, aromatic halides (aryl halides), aromatic heterocyclic compounds, aromatic hydrocarbons (arenes), and aromatic nitro compounds (aryl nitro compounds).
As used herein, the terms xe2x80x9caliphaticxe2x80x9d and xe2x80x9caliphatic compoundsxe2x80x9d refer to compounds which comprise carbon atoms in chains, rather than the ring structure of aromatic compounds. It is intended that these aliphatic moieties will be bound to additional elements in some embodiments.
The terms xe2x80x9cresistantxe2x80x9d and xe2x80x9crefractoryxe2x80x9d used in reference to xe2x80x9cresistant mutantsxe2x80x9d of HIV and/or HIV RT, refer to the ability of some HIV RTs to function in the presence of compounds that are inhibitory to the RT of wild-type HIV. This resistance may result from any number of mutations, including but not limited to conformational changes in the RT structure, as well as to the configuration of the RT bound to its substrate.
The term xe2x80x9cmixturexe2x80x9d refers to a mingling together of two or more substances without the occurrence of a reaction by which they would lose their individual properties. The term xe2x80x9csolutionxe2x80x9d refers to a liquid mixture. The term xe2x80x9caqueous solutionxe2x80x9d refers to a solution that contains some water. In many instances, water serves as the diluent for solid substances to create a solution containing those substances. In other instances, solid substances are merely carried in the aqueous solution (i.e., they are not dissolved therein). The term aqueous solution also refers to the combination of one or more other liquid substances with water to form a multi-component solution.
The terms xe2x80x9csamplexe2x80x9d and xe2x80x9cspecimenxe2x80x9d in the present specification and claims are used in their broadest sense. On the one hand, they are meant to include a specimen or culture. On the other hand, they are meant to include both biological and environmental samples. These terms encompass all types of samples obtained from humans and other animals, including but not limited to, body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, and saliva, as well as solid tissue. These terms also refer to swabs and other sampling devices commonly used to obtain samples for culture of microorganisms.
Biological samples may be animal, including human, fluid or tissue, food products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, disposable, and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.
As used herein, the term xe2x80x9cculturexe2x80x9d refers to any sample or specimen which is suspected of containing one or more microorganisms. xe2x80x9cPure culturesxe2x80x9d are cultures in which the organisms present are only of one strain of a particular genus and species. This is in contrast to xe2x80x9cmixed cultures,xe2x80x9d which are cultures in which more than one genus, species, and/or strain of microorganism are present.
As used herein, the term xe2x80x9corganismxe2x80x9d is used to refer to any species or type of microorganism, including but not limited to viruses. In particular, the term is used in reference to RNA viruses, such as the retroviruses. In preferred embodiments, the organism of interest is HIV. In particularly preferred embodiments, the organism of interest is HIV-1.
The term xe2x80x9cparenterallyxe2x80x9d refers to administration to a subject through some means other than through the gastrointestinal tract or the lungs. The most common mode of parenteral administration is intravenous. However, other modes of parenteral administration include, but are not limited to, intramuscular, intradermal, intrathecal, intranasal and subcutaneous administration.
As used herein, the term xe2x80x9cpharmaceutical compositionxe2x80x9d refers to compositions composed of one or more pharmaceutically acceptable diluents, excipients or carriers. As used herein, the phrase xe2x80x9cpharmaceutical preparation suitable for parenteral administrationxe2x80x9d refers to a solution containing compound in a pharmaceutically acceptable form for parenteral administration. The characteristics of the form will depend on a number of factors, including the mode of administration. For example, a preparation for intravenous administration will often comprise compound dissolved in normal saline or sterile water for injection. Of course, the pharmaceutical preparations of the present invention are not limited to those diluents; indeed, other components or diluents known in the field of pharmaceuticals and pharmacy are within the scope of the present invention. The pharmaceutical preparation may contain diluents, adjuvants and excipients, among other components, provided that those additional components neither adversely effect the preparation (e.g., they do not cause degradation of the compound) nor the recipient (e.g., they do not cause a hypersensitivity reaction).